Nucleic acid aptamers methods

Prerequisites to this approach are methods of incorporating non-natural moieties at predetermined positions in the biopolymer. This volume is meant to serve as a source in this respect describing the state of the art of some major lines of attack with this goal in mind. As a modern alternative, the creation of novel catalysts by directed evolution of nucleic acid aptamers is included. In this case, too, it is of prime importance to learn about the structural details which cooperate to bring about the catalytic function underlying the selection process. 

SELEX is a widely used technique for screening of aptamers which are nucleic acid ligands. According to this method, a pool of DNA with a random sequence region attached to a constant chain is constituted by amplification then transcribed to RNA. RNA pool is separated according to the affinity of RNA molecules to a target protein. DNA molecules obtained by reverse transcription from retarded RNA molecules are amplified and the cycle is repeated. 

A more recent application of redox labeled ODNs is redox-active aptamers that exploit molecular recognition between the aptamer and a target analyte. Briefly, aptamers are functional nucleic acids that selectively bind to a variety of targets. Due to a well-defined three-dimensional structure, aptamers can achieve selectivity comparable to that of antibodies but are readily accessible taking advantage of well-known nucleic acid chemistry, polymeric chain reaction and contemporary separation methods, followed by aptamer selection from random pools of nucleic acids (DNA or RNA) by in vitro selection process called systematic evolution of ligands by... 

The formation of aptamer-substrate complexes was also followed by the use of redox-active intercalators73 (Fig. 12.18d). A nucleic acid hairpin structure that contained in its single-stranded loop the antithrombin base sequence was assembled on a Au electrode, and methylene blue was intercalated as a redox label in the double-stranded stem of the hairpin structure. The hairpin was, then, opened in the presence of thrombin, by generating the respective G-quadruplex-thrombin complex, and as a result, the redox label was removed from the nucleic structure, showing a decrease in the voltammetric response with the increase in the concentration of thrombin. This method enabled the analysis of thrombin with a detection limit that corresponded to... 

Since the discovery of catalytic nucleic acids and the development of methods used to generate novel species in vitro [221,222], a wide range of applications has been devised—with many implemented in an immobilized format. Like aptamers, the functionality of these nucleozymes is generally not diminished by attachment to a surface, either through affinity [223] or covalent immobilization [224], making the transition to immobilized strategies relatively straightforward. 

The popularity of aptamer-based assays has risen over the last several years in proteomics (25). Typical aptamers are single-stranded nucleic acids that can recognize target molecules. The general basis is similar to immuno-based methods. These aptamers are found by screening large libraries of nucleic acid sequences. This technology has been applied to several proteome-level detections (26-29). 

There are two general steps to evolving a catalytic RNA. First, from a pool of sequence variants there must be a screening protocol that separates active RNA with the desired trait from others without it. Second, the genome of the survivors must be copied and amplified via DNA intermediates prior to the next round of selection. The process is iterative, and heavily reliant on the ease of nucleic acid copying by PCR amplification. Thereafter, there are two methods of selection, direct or indirect. In a direct selection, there is chemical transformation of the catalytic RNA during the selection step. In an indirect selection, an RNA aptamer approach is taken but RNA aptamers are identified and evolved to bind to a transition state in the reaction that is desired to be catalysed (this indirect method is similar to the strategy used to develop catalytic antibodies - see Section 10.5). 

The positions that remain unmodified at the end of the in vitro selection procedure (e.g., the purine residnes in a selection carried out with 2 -fluoro-pyrimidine triphosphates) can be modified post-SELEX for further optimization of the aptamers. A systematic stndy of the 64 variants of the six-membered apical loop of an anti-TAR aptamer led to the identification of locked nucleic acid/2 -0-methyl chimeras fully resistant to nncleases that displayed anti-HIV-1 properties in a cell cnlture assay (Di Primo et al., 2007). Identification of the few residues that cannot be modified in an RNA aptamer can be carried ont by chemical interference, a method nsed to identify chemical variants of the aptamer originally selected. Snch an approach led to the synthesis of a modified anti-HIV-1 reverse transcriptase in which all bnt two of the positions of the RNA aptamer were snbstitnted by 2 -0-methyl residnes (Green et al., 1995). This was also the case for the aptamer nsed for age-related macnla degeneration in hnman beings (Ruckman et al., 1998). 

Mnrphy, M. B., Fuller, S. T., Richardson, P. M., Doyle, S. A. (2003). An unproved method for the in vitro evolution of aptamers and applications in protein detection and purification. Nucleic Acids Res 31, ellO. 

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